Project summary Cyanide poses a significant threat to human health. Mass exposures to cyanide through industrial accidents or terror attacks would have devastating effects without effective and easily administered antidotes. A few compounds with proven cyanide antidotal activity already exist, but they suffer from low potency and difficult routes of administration. Potent and highly efficacious cyanide countermeasures are still needed, and methods that enable discovery of truly novel countermeasures with novel mechanisms of action are particularly attractive. The proposed center of excellence will discover and develop cyanide countermeasures that are highly potent and function through novel mechanisms. Project 1 employs a validated, large-scale chemical screen to discover compounds that protect zebrafish from cyanide toxicity. Many of these initial hits will not possess sufficient potency or selectivity to be strong preclinical drug leads. In Project 2, we will use medicinal chemistry methodologies to optimize the potency of the hits discovered in Project 1. Optimized compounds will then be profiled to determine their stability, bioavailability, toxicity, and metabolomic effects. These experiments will enable us to transform screening hits into potent drug leads with acceptable pharmacokinetic properties and minimal toxicities. The best of the optimized drug leads will be delivered to Project 3 for further efficacy testing in mammals. Specifically, we propose the following aims: Aim 1. To optimize the potency of novel cyanide countermeasures. Aim 2. To profile the pharmacological properties of optimized candidate countermeasures. By completing these aims, we will provide an essential bridge between the high-throughput discovery effort of Project 1 and the validated efficacy models of Project 3. The compound optimization process outlined herein takes advantage of several innovations (in vivo SAR studies, high-throughput zebrafish toxicology, metabolomics) to make the process much faster and more cost effective than the traditional drug development pathway. Together, the projects and cores will deliver truly new countermeasure classes with new mechanisms of action that transform our ability to respond to cyanide threats.